Abstract
Background/Aim: Acute exogenous lipoid pneumonia (AELP) is a rare disorder caused by intake of lipid formulations and is often underdiagnosed. Meanwhile, the mechanism of AELP is still underlying. MCC950, was previously found to significantly suppress the release of inflammatory cytokines IL-18 and IL-1β. However, the effect of MCC950 on AELP induced by sewing machine oil has not been reported. Materials and Methods: The NLRP3, NF-
B p65, caspase-1 and IL-1β expression in lung tissues were compared between a rat model of AELP and control rats using western blotting and real-time quantitative assay. Moreover, haematoxylin and eosin (H&E) staining was performed to elucidate the mechanisms by which MCC950 ameliorates sewing machine oil-induced AELP in vivo. Results: MCC950 reduced the expression of NF-
B p65 in the lung samples of the treatment group and further down-regulated the NLRP3 and caspase-1 levels while inhibited the production of IL-1β. Besides, decreases in inflammatory cell infiltration in the lung were shown using H&E staining. Conclusion: MCC950 ameliorates sewing machine oil-induced acute exogenous lipoid pneumonia in rats through inhibition of the NF-
B/NLRP3 inflammasome pathway.
Acute exogenous lipoid pneumonia (AELP) is a non-infectious disease caused by intake of lipid formulations and is generally underdiagnosed. Inflammatory reaction in AELP depends on oil exposure route and degree, and biochemical characteristics of oil, like volatility and viscosity (1). One of the earliest reports on AELP in Canadian children with nasopharyngeal injections of oil was based on autopsy findings (2). Usually, AELP is unrecognized, because the symptoms are nonspecific and often is confused with infectious or other lung diseases according to roentgenographic findings. Unusual manifestations of AELP are reported including cavitating lung and pneumothorax, which may result from high volume kerosene exposure (3). To date, treatment of AELP has not been standardized, evidence-based guidelines are missing.
Discontinuation of lipid exposure is the most used intervention. Meanwhile, corticosteroids in severe cases, antibiotics, and therapeutic lung lavage are also effective and safe therapies (4, 5). Patients, who intake massive lipids once in a short time, may develop severe, life-threatening respiratory failure and ventilator support may be required (6). Furthermore, the oil cannot be emulsified by lung lipases and will be engulfed by alveolar macrophages localized in the alveolar wall, eliciting severe inflammatory response (7). Therefore, to explore the molecular variations in AELP helps to standardize the diagnosis and therapy of AELP.
Inflammasomes have been proved to participate in inflammation in both infectious and non-infectious diseases. NLRP3 is the best characterized NOD-like receptor (NLR) family member pyrin domain-containing protein 3 (NLRP3) inflammasome. It is an oligomeric complex produced during the innate immune response to pathogen-, host- and environmental-derived factors (8). Recognition of damage or pathogen associated molecular patterns (DAMPs/PAMPs) leads to activation of nuclear factor kappa B (NF-
B) signalling pathway and subsequently up-regulates transcription of inflammasome-related mediators, including NLRP3, pro-IL-1β, and pro-IL-18. Then the inflammasome-adaptor protein ASC is recruited and leads to caspase-1 autocatalytic activation. This result can promote the maturation of pro-inflammatory cytokines interleukin (IL)-1β, and interleukin (IL)-18 (9, 10). Mounting evidence has shown the important effect of the NLRP3 inflammasome on all kinds of lung diseases. NLRP3 inflammasome activation triggers a series of responses to lung fibrosis induced by the fine particulate matter (PM2.5). MCC950 is known to be a specific small molecule inhibitor that selectively blocks the activation of the NLRP3 inflammasome (11) without inhibiting the AIM2, NLRC4 or NLRP1 inflammasomes (12). Temporal administration of MCC950 reduced influenza A disease severity through decreased cytokine production and lung cellular infiltrates (13). In our study, a sewing machine oil-induced rat model AELP was established to examine the protective effect of MCC950. We suggest that MCC950 plays an important role in the negative regulation of NF-
B p65-NLRP3-IL-1β pathway and may be a novel therapy for AELP.
Materials and Methods
Model validation of acute exogenous lipoid pneumonia.
Experimental animals. All procedures were executed following the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication NO. 85-23, revised in 1996). Ethical approval of the protocol was obtained from the Ethics Committee (Protocol number: wydw2022-0620). Male Sprague-Dawley rats (body weight 180-220 g; 3-4 weeks of age) were obtained from the Wenzhou Medical University, Zhejiang China (SCXK 2018-0017). Animals were housed in a room with controlled at temperature (20-22°C) and a relative humidity of approximately 40% and a 12:12h light/dark cycle.
Establishment and validation of the rat model. The rat model of AELP was generated following the method established by G.G. Yampara (14). Rats were divided into four groups of 5 animals each as follows: Control group, intake of 1.0 ml/kg normal saline (NS, NS group); AELP1 group (intake of 0.5 ml/kg sewing machine oil); AELP2 group (intake of 0.75 ml/kg sewing machine oil) and AELP3 group (intake of 1.0 ml/kg sewing machine oil). The effects of various concentrations of sewing mineral oil on rats are shown in Figure 1A and B. Twenty -four hours after intake, the survival rate was 80% in the AELP1 group. As all rats in AELP2 group and AELP3 group died within 24 h, we examined the effect 0.5 ml/kg in subsequent experiments. The rats were anesthetized with intraperitoneal injection of 0.3% sodium pentobarbital 24 h after had been administered with the oil. Chest X-ray for rat: The rats administered anaesthesia were positioned in a supine position for X-ray imaging. The X-ray parameters used were 65 kV and 16 mAs.
Effect of various concentrations of sewing mineral oil on rats. (A) Survival curves for rats in each group. The mortality rate was higher in the AELP2 (0.75 ml/kg) and AELP3 (1 ml/kg) groups than that in AELP1 (0.5 ml/kg) group. (B) Lung histopathology of rats in the NS and AELP1 groups at 24 h after inhalation.
In vivo AELP model and treatment. In order to induce inflammation in rats, the animals were orally intubated under non-invasive direct vision and administered 0.5 ml/kg sewing machine oil as previously described (15). Each group consisted of 5 as follows 1) NS group was intraperitoneally injected with a dose of 0.3 ml NS at 12, 24, and 48 h after model establishment; 2) AE group (intake of 0.5 ml/kg sewing machine oil) was intraperitoneally injected with the same dose of NS at 12, 24 and 48h after inhalation, 3) MCC950 group was administered the oil and injected intraperitoneally with MCC950 at a dose of 10 mg/kg at 0.5h before inhalation and 12, 24, and 48 h after exposure to the oil; 4) Pyrrolidine dithiocarbamate (PDTC) group was intraperitoneally injected with PDTC at a dose of 100 mg/kg at 1 h before inhalation and 12, and 24 h exposure to the oil. To allow the liquid to enter the lungs and distribute evenly, the rats were rotated back and forth 10 times after exposure. Finally, samples of the lung tissue, blood and bronchoalveolar lavage fluid (BALF) were collected to further perform pathological and biochemical characterization. This was done under anesthesia.
Inhibitors of NLRP3 and NF-
B. The NLRP3 inhibitor MCC950 was obtained from MedChem Express (No.256373-96-3, Middlesex County, NJ, USA). PDTC, selective inhibitor of NF-
B, was purchased from Proteintech Group (Hubei, PR China). Doses of drug determined to be harmless were determined through cytotoxicity and viability assays.
Haematoxylin and eosin (H&E) staining. The right lower lobe of the lung tissues was harvested on day 1 and 3 after exposure, fixed using 4% paraformaldehyde solution and embedded in paraffin. Pathological changes in lung tissues were observed using haematoxylin-eosin (HE) staining and light microscopy. Lung injury was assessed based on the microscopic examination of slices stained with H&E dye and a five-point numeric scores (16). Each item was scored on a 5-point scale as follows: 0=minimal damage, 1+=mild damage, 2+=moderate damage, 3+=severe damage, and 4+=maximal damage.
Wet-to-dry weight ratio (W/D). The wet-to-dry weight ratio of the right upper lobe lung tissue was compared. Samples of the lung tissue were examined to observe the lung tissue oedema and histological changes. The lobes were washed using NS and completely dried for 24 h at 80°C.
Reverse-transcription polymerase chain reaction (RT-PCR). RNA was extracted from the lung tissues using an RNA isolation kit (Takara Biomedical Technology Co., Ltd. Beijing, PR China) according to manufacturer’s instructions. The total RNA was reverse transcribed into cDNA using the FastQuant RT kit (Shanghai TianShai Biotech Co., Ltd, Shanghai, PR China) in 10 μl reaction volumes containing 2μl of total RNAs. Then Real-time qPCR was executed using a Real-Time PCR System (BIORAD, Hercules, CA, USA) and the cycling program: 95°C for 60 s followed by 40 cycles (95°C for 15 s, 60°C for 15 s, and 72°C for 45 s). The Actb gene (encoding b-actin) was used as an internal control. The primer sequences for the RT-qPCR assay were synthesized by Sangon Biotech Co., Ltd. (Shanghai, PR China) and shown in Table I.
Primer sequences of target mRNA.
Western blotting. Proteins were extracted from lung tissues using RIPA buffer (Beyotime, Shanghai, PR China). Primary antibodies were incubated overnight at 4°C and secondary antibody (1:2,000) was incubated for 1 h. The primary antibodies were mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (1:1,000), anti-NF-
B p65 (1:1,000), anti-caspase-1 (1:1,000), anti-IL-1β (1:1,000) anti-NLRP3 (1:1,000). Horseradish peroxidase-conjugated mouse anti-rabbit IgG-HRP (1:2,000) was used as the secondary antibody. The immunoreactive bands were detected using enhanced chemiluminescence. The NF-
B p65 and caspase-1 antibodies were obtained from CST (Boston, MA, USA), the secondary antibody and IL-1β antibody were from Santa Cruz (Santa Cruz, CA, USA), NLRP3 antibody was from Beyotime.
Enzyme linked immunosorbent assay (ELISA). The levels of IL-1β and IL-18 in BALF samples were measured using ELISA Kits (Shanghai Boyun Bio-tech Co. Ltd, Shanghai, PR China). The assays were performed following the manufacturer’s instructions.
Statistical analysis. The grouped data are presented as mean±SD of three replicates and evaluated statistically using One-way ANOVA with post hoc correction (Tukey’s post-comparison test). Analytical figures were drawn and calculated using GraphPad Prism (Version 9, San Diego, CA, USA). Comparing 2 values (two-tailed, two-sample equal variance), we used a Student’s t-test. A p-value <0.05 was considered statistically significant.
Results
MCC950 treatment alleviates lung tissue injury. To investigate the effects of MCC950, AELP model was induced by sewing machine oil in rats. As shown in Figure 2A, the pulmonary tissue showed varying degrees of swelling. Lung tissue in the NS group was slightly swollen, while in the AE group, the lung showed obvious swelling and bleeding. Focal haemorrhage scattered on the surface of the lung tissue was ameliorated in the MCC950 and PDTC groups. As expected, the lung interstitium and the alveolar cavity in the NS group were infiltrated on the 1st and 3rd days with some neutrophils. However, higher infiltration of inflammatory cells and erythrocytes was observed in the pulmonary interstitium in the AE group, compared with NS group. However, in the MCC950 and PDTC groups this was occasionally observed and was lower than that in the AELP group (Figure 2B). Histological scores were also evaluated to quantify the damage (Figure 2C). The AE group scored higher than the NS group, whereas the MCC950 and PDTC groups scored lower than the AE group. The above results indicate that MCC950 significantly alleviates lung tissue injury.
MCC950 treatment alleviates lung tissue injury. (A) Varying degrees of pulmonary tissue swelling on the 3rd day following intake. The pulmonary tissue of the AE group shows obvious swelling, and bleeding spots are observed on the surface. After MCC950 or PDTC treatment, the swelling degree of the lung tissue was improved, and bleeding was reduced. (B) Haematoxylin and eosin staining of lung parenchyma tissue on day 1 and 3 after intake. Inflammatory cell and erythrocyte infiltration in the pulmonary interstitium are shown on the 1st and 3rd day in different groups. (C) Histological scores evaluated as described in the Materials and Methods section. Results are presented as mean±SEM values; *p<0.05, ***p<0.01. (D) The wet/dry (W/D) ratio was used to determine the pulmonary oedema. All data are presented as the mean±SD. *p<0.05, compared with NS group; #p<0.05, compared with AE group.
Wet/Dry ratio. The wet/dry (W/D) ratio was used to evaluate the pulmonary oedema. Compared to the NS group, the W/D ratio of the AE group increased significantly on the 1st day and continued increasing up to 3 days. However, following treatment with MCC950 and PDTC, the W/D ratios increased greatly on day 1, but decreased to levels comparable to those of the control group on day 3, indicating the protective effect of MCC950 and PDTC on pulmonary oedema (Figure 2D).
Effects of MCC950 on the expression of NF-
B, NLRP3, caspase-1 and IL-1β in lung tissue. Based on the protective effect of MCC950 (the chemical structure in Figure 3A) and PDTC, we hypothesized a positive correlation between increased W/D ratio and activation of NLRP3 inflammasome during AELP. Western blot analyses were performed to monitor the changes in relevant signalling molecules. As illustrated in Figure 3B and C, the levels of NLRP3, NF-
B, Caspase-1, and IL-1β increased significantly both on day1 and day 3 in the AE group and all were downregulated upon treatment with MCC950 and PDTC. These results show that NLRP3 inflammasome is activated following exposure of the lungs to exogenous oil.
Effects of MCC950 on AELP. The expression of NF-
B, NLRP3, caspase-1, and IL-1β was analysed. (A) The chemical structure of MCC950. (B) Representative images of western blot results. (C) Quantitative densitometric analysis of proteins. Treatment with MCC950 decreased NF-
B, NLRP3, caspase-1 and IL-1β expression. Values are means±SD, *p<0.05 compared with NS group and #p<0.05 compared with AE group.
MCC950 down-regulated NF-
B, NLRP3, caspase-1 and IL-1β in the lung tissue. The transduction pathway of the NF-
B/NLRP3/IL-1β has been considered as essential for an early inflammatory response (17, 18). In our study, RT-qPCR was performed to determine the levels of downstream-signalling molecules. Hence, the expression of NF-
B and NLRP3 were examined to illustrate whether the anti-inflammatory effects of MCC950 contribute to the amelioration of AELP. As showed in Figure 4, the AE group significantly higher NF-
B, NLRP3, caspase-1and IL-1β levels (p<0.05). Whereas MCC950 effectively treatment suppressed the expression of NLRP3 (p<0.05), NF-
B (p<0.05), caspase-1 (p<0.05) and IL-1β levels (p<0.05).
The mRNA levels of NF-
B, NLRP3, caspase-1 and IL-1β were determined using quantitative real-time PCR. All data are presented as the mean±SD. There is an increase in the AE group compared with the NS group. *p<0.05, ***p<0.01.
MCC950 down-regulated the levels of IL-1β and IL-18 in BALF. In our study, the expression of IL-1β and IL-18 in BALF were examined to clarify the effect of MCC950 on the NF-
B/NLRP3/IL-1β pathway. As depicted in Figure 5, the AE group showed significant up-regulation of IL-1β and IL-18 levels (p<0.01). However, the expression of IL-1β and IL-18 (p<0.01) decreased in the MCC950 treatment group. The findings demonstrated that the inhibition of the NF-
B/NLRP3/IL-1β signalling pathway was involved in the protection against AELP.
The expression of IL-1β and IL-18 in BALF was measured using ELISA. All data are presented as the mean±SD. *p<0.05, ***p<0.01.
Discussion
Laughlen (2) first reported four cases of a rare form of pneumonia induced by the intake of fatty substances in 1925 and named it exogenous lipoid pneumonia (ELP). Following cases indicated that ELP mainly results from the intake of mineral, vegetal or animal oily substances. Excessive inflammation can cause acute respiratory failure or death, particularly following intake of high-dose oil. However, the underlying mechanisms of excessive cellular infiltration and cytokine storm related to AELP are poorly understood. Lipidomic analysis using conventional gas chromatography combined with liquid chromatography (LC)/mass spectrometry (MS) enabled a comprehensive and quantitative analysis of lipids (19, 20). Lipid-loaded macrophages are found in respiratory tract specimens of patients including sputum, bronchoalveolar lavage fluid, or fine-needle aspiration cytology/biopsy from pulmonary lesions and can be used in the diagnosis AELP (21). There are no evidence-based treatment guidelines, so the treatment of the disease is not standardized. Common interventions consist of discontinuation of lipid exposure, antibiotics, corticosteroids, and therapeutic lung lavage in critical patients (22). It is often necessary to perform specific stainings to reach the diagnosis (23, 24).
Several signalling pathways are related to inflammatory response, and nuclear factor-
B (NF-
B) is widely perceived a critical for the disease. NF-
B interacts with I
B to be a silent state and has no effect on the expression of downstream genes in normal conditions. However, following stimulation, I
B is phosphorylated and NF-
B is released and activated to regulate the levels of inflammatory cytokine genes. Activated NF-
B regulates cell proliferation and apoptosis, and the expression of inflammatory cytokines such as tumour necrosis factor (TNF)-α, IL-1β, IL-6 and IL-8 (25). Accumulating evidence suggests that the NLRP3 inflammasome has an effect on acute lung injury (26). The NLRP3 inflammasome is a multiprotein complex found in macrophages, dendritic cells, and other nonimmune cells (27). NF-
B signalling is also involved in the activation of NLRP3 inflammasome (28, 29), and budesonide can inhibit the expression of NF-
B (30). NLRP3 deletion has shown protective effects on septic cardiomyopathy and suppresses NF-
B signalling in sepsis-induced cardiomyopathy (31). Furthermore, current studies have demonstrated that activated NF-
B pathway could function as an upstream activator of inflammasome including NLRP3, ASC and serine protease caspase-1. Herein, we found that there was increased expression of NF-
B aggravating lung tissue damage in the AE group. However, treatment with PDTC reduced the level of NLRP3 and IL-1β in lung samples. These results demonstrate that NF-
B p65 inhibitors could alleviate oil-induced pulmonary injury.
IL-1β acts as a proinflammatory cytokine activates, promotes the differentiation of lymphocytes, regulates infiltration of inflammatory cells, leads to chemotaxis and induces the expression of other inflammatory cytokines, regulates the function of epithelial cells, and might result in tissue injury by mediating inflammation (32). The NLRP3 inflammasome regulates the cleavage and maturation of the proinflammatory factor IL-1β. The NLRP3 inflammasome is activated through priming and assembly of the multimeric complex. The priming procedure results in the up-regulation of the transcription of NLRP3, IL-1β and IL-18, and promotes NLRP3 post-translational silencing (33). It was widely known that NF-
B plays a role upstream of NLRP3 (34). Whereas, over-expressed NLRP3 also contributes to the expression of IL-1β and IL-18, and many studies showed that IL-1β can stimulate the activation of NF-
B signalling pathway through a feedback loop (35-38). The up-regulated IL-1β production might lead to the lung injury. Therefore, we developed an AELP model to gain insight into the mechanism of NF-
B–NLRP3–IL-1β pathway in sewing machine oil-induced AELP. Compared to NS group, the lung damage in the AE group was aggravated sharply indicating that the AELP model was successfully established. MCC950 has recently been shown to be an NLRP3 inhibitor (39). We found that MCC950 significantly alleviated lung inflammation in the AE group. MCC950 inhibited the inflammatory reaction by reducing the expression of NF-
B, NLRP3, caspase-1 and IL-1β (Figure 6). The levels of NLRP3, caspase-1 and IL-1β can be increased by increased NF-
B expression, which can further promote the occurrence and aggravate inflammation. Therefore, it was shown that the expression of NLRP3 is regulated by NF-
B. It was also suggested that AELP can result in increased IL-18 level through the NF-
B–NLRP3–IL-1β signalling pathway thereby aggravating lung injury.
The mechanism of action of MCC950. MCC950 suppressed inflammation in sewing machine oil-induced pulmonary injury through NF-
B/NLRP3 signalling pathway.
Nevertheless, the expression of NF-
B, NLRP3 and IL-1β in lung samples of the AE group were significantly increased compared to those of the NS control group. Their expression decreased on the third day in the MCC950 group. Our findings showed that MCC950-mediated inhibition of NLRP3 inflammasome activation may be critical for restricting the pathology of AELP. Cognizant to this, further studies are required to explore this mechanism.
Conclusion
MCC950, as a specific NLRP3 inhibitor, can ameliorate pulmonary inflammation in young rats induced by the intake of sewing machine oil through the inhibition of the NF-
B–NLRP3–IL-1β signalling pathway. Targeting the inflammasome with MCC950 is a potential promising approach for the treatment of AELP.
Footnotes
Authors’ Contributions
WDY and HMW designed the work, acquired, and analyzed data, and participated in writing the manuscript; DSL, ARH, ZWX, and XGH acquired and analyzed data and revised the manuscript; and YMJ contributed to the concept and design of the work, reviewed, and revised the manuscript.
Funding
This work was supported by Wenzhou Public Welfare Science and Technology Projects [grant no. Y20210005].
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study. The funding agencies had no role in the design, execution, interpretation, or writing of the study.
- Received June 22, 2023.
- Revision received July 27, 2023.
- Accepted July 31, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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